Melatonin pathway genes are associated with progressive subtypes and disability status in multiple sclerosis among Finnish patients

Journal of Neuroimmunology 250 (2012) 106–110

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Melatonin pathway genes are associated with progressive subtypes and disability status in multiple sclerosis among Finnish patients Renuka Natarajan a, Elisabet Einarsdottir b, Asko Riutta c, Sanna Hagman a, Minna Raunio d, e, Nina Mononen f, Terho Lehtimäki f, Irina Elovaara a, d,⁎ a

Neuroimmunology Unit, School of Medicine, University of Tampere, Tampere, Finland Department of Medical Genetics and Research Program for Molecular Medicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland Department of Pharmacological Sciences, School of Medicine, University of Tampere, Tampere, Finland d Department of Neurology, Tampere University Hospital, Tampere, Finland e Department of Neurology, Kanta-Häme Central Hospital, Hämeenlinna, Finland f Fimlab Laboratories, Tampere University Hospital and Department of Clinical Chemistry, School of Medicine, University of Tampere, Tampere, Finland b c

a r t i c l e

i n f o

Article history: Received 26 January 2012 Received in revised form 4 May 2012 Accepted 22 May 2012 Keywords: Multiple sclerosis Melatonin pathway genes Single nucleotide polymorphisms (SNPs) TPH2 MTNR1B Expanded Disability Status Scale (EDSS)

a b s t r a c t In this study we investigated the relationship between melatonin pathway and multiple sclerosis (MS) in a high-risk Finnish population by studying the single nucleotide polymorphisms (SNPs) in the genes coding for critical enzymes and receptors involved in the melatonin pathway. A total of 590 subjects (193 MS patients and 397 healthy controls) were genotyped for seven SNPs in four genes including tryptophan hydroxylases (TPH)1 and 2, arylalkylamine N-acetyltransferase (AANAT) and melatonin receptor 1B (MTNR1B). An overrepresentation of T allele carriers of a functional polymorphism (G-703T, rs4570625) in the promoter region of TPH2 gene was observed in the progressive MS subtypes. The haplotype rs4570625rs10506645TT of TPH2 gene was associated with the risk of severe disability in primary progressive MS (PPMS), while haplotype rs4570625-rs10506645TC appeared to be protective against disability in secondary progressive MS (SPMS). In the MTNR1B gene, the haplotype rs10830963-rs4753426GC was associated with the risk of SPMS, whereas another haplotype rs10830963-rs4753426GT showed an association with the risk of PPMS. These data showing the association of polymorphisms in the TPH2 and MTNR1B genes with the progressive subtypes of MS and disability suggest dysregulation in melatonin pathway. Melatonin pathway seems to be involved in disease progression, and therefore its potential effects in overcoming MSrelated neurodegeneration may be worth evaluating in future clinical trials. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS) that develops in genetically susceptible individuals and may require additional environmental triggers causing immune dysregulation leading to neuroinflammation and degeneration (Hauser and Oksenberg, 2006). MS has an increasing prevalence with increasing latitudes and the disease affects approximately 0.1% of the population of the northern hemisphere (Hafler, 2004). Among environmental factors, insufficient exposure to sunlight has been shown to be associated with the development of MS. Prior research has focused on the ultraviolet radiation of sunlight, its ability to influence melatonin synthesis, and an association with vitamin D metabolism (Smolders et al., 2008; Mehta, 2010). Sunlight has been reported to have both direct and indirect effects ⁎ Corresponding author at: University of Tampere, School of Medicine, Neuroimmunology Unit and Department of Neurology, Lääkärinkatu 1, Arvo Building, Room b322, 33014, Finland. E-mail address: irina.elovaara@uta.fi (I. Elovaara). 0165-5728/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2012.05.014

on melatonin biosynthesis (Schwarz, 2005; Hauser and Oksenberg, 2006). Since Finland is located at high latitude with a prolonged period of darkness during the winter and has one of the highest rates of MS in the world (Sumelahti et al., 2001), it is appropriate to investigate the relationship between the melatonin pathway and MS in such a high-risk Finnish population. Melatonin, a hormone contributing to a wide array of physiological functions, is secreted by the pineal gland specifically at night (Simonneaux and Ribelayga, 2003). The biosynthesis of melatonin begins with the hydroxylation of L-tryptophan into serotonin by tryptophan hydroxylases (TPH) 1 and 2. Subsequently, serotonin is N-acetylated by arylalkylamine N-acetyltransferase (AANAT) to Nacetylserotonin, which is converted to melatonin by hydroxyindoleO-methyltransferase (Chattoraj et al., 2009). Melatonin partly exerts its physiological effects by acting on its G-protein coupled membrane receptors (MT1 and MT2) or by the nuclear receptors belonging to the RZR/ROR orphan receptor subfamily (Dubocovich et al., 2003). In recent years, the antioxidant property of melatonin via free radical scavenging mechanism has been considered to protect against neuronal cell death. These studies provide a rationale for

R. Natarajan et al. / Journal of Neuroimmunology 250 (2012) 106–110

trials evaluating the possibility of beneficial effects of melatonin in neurodegenerative disorders which may also be adapted for MS (Sharma et al., 2006; Reiter et al., 2009). In MS, the degree of motor disability and chronic progressive course of the disease seems to correlate with the depletion of serotonergic function (Sandyk, 1998; Markianos et al., 2009). Additionally, it has also been shown that the production of melatonin is lowered and phase-shifted in MS patients with depressive symptoms that have been explained by pineal gland calcification and hypothalamic dysfunction (Sandyk and Awerbuch, 1993; Akpinar et al., 2008). Furthermore, the inverse association detected between nocturnal melatonin secretion and duration of clinical symptoms suggests that the function of the pineal gland declines with the progression of MS (Sandyk and Awerbuch, 1994). The association detected between melatonin pathway genes with various other disorders has been studied (see supplementary Table 1), but until now no single study has addressed their association with MS. In this exploratory study based on a high-risk Finnish population, we examined the polymorphisms in the genes coding for critical enzymes and receptors involved in melatonin biosynthesis and analyzed whether these polymorphisms constitute risks for the MS disease and the development of progressive disability. For this purpose, we selected four genes, namely tryptophan hydroxylase (TPH I and II) aryl-alkylamine N-acetyltransferase (AANAT) and melatonin receptor 1B (MTNR1B) essential for the synthesis and function of melatonin and studied their single nucleotide polymorphisms (SNPs) that have been shown to affect the melatonin pathway in other diseases (Supplementary Table 1). We analyzed allele, genotype and haplotype association to inflammatory (RRMS) and degenerative subtypes (SPMS and PPMS) of MS and neurological disability. 2. Patients and methods 2.1. Study population This case–control genotyping study comprised a total of 590 subjects. One hundred and ninety three patients (126 females, 67 males), including 109 relapsing remitting MS (RRMS), 51 secondary progressive MS (SPMS) and 33 primary progressive MS (PPMS) patients took part in this study. The diagnosis of MS was based on McDonald's Criteria (McDonald et al., 2001; Polman et al., 2005). As inflammatory changes are characteristic for RRMS and degenerative changes for SPMS and PPMS, we termed the RRMS group the inflammatory MS group (n = 109) and combined SPMS and PPMS groups as progressive MS group (n = 84). Three hundred and ninety seven (397) healthy subjects with similar range of age were included as controls. All MS patients underwent neurological evaluation including the determination of Expanded Disability Status Scale (EDSS) score (Kurtzke, 1983) (Table 2). The progression index was calculated as: disability score divided by the duration of the disease. The study was approved by the Ethics Committee of the Tampere University Hospital and all subjects gave their informed consent. The patient groups were divided on the basis of their EDSS scores to assess the association of alleles and haplotypes with neurological disability in both inflammatory and progressive MS: those with EDSS scores less than 4 were considered mildly disabled, while those with EDSS scores higher than 4 were more severely disabled. 2.2. Genotyping Three genes (TPH1, TPH2, and AANAT) in melatonin biosynthesis pathway as well as a membrane receptor gene (Melatonin receptor 1B, MTNR1B) essential for the function of melatonin were genotyped. The following SNPs: TPH1 (rs1800532 and 10488982), TPH2 (rs10506645 and rs4570625), AANAT (rs12942767) and MTNR1B (rs4753426 and rs10830963) were chosen based on prior evidence

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of their possible role in the melatonin pathway (Supplementary Table 1). Genomic DNA was extracted from buffy coats using the QIAamp DNA Blood Minikit and automated biorobot M48 extraction (Qiagen, Hilden, Germany). Genotyping was performed using Taqman SNP Genotyping Assays C_8940793_10, C___2645675_10_, C___30277975_20, C___226207_10, C___31556625_20, C___289583_10, C___3256858_10 and the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, California, USA). Random duplicates and control samples were run in parallel with unknown DNA samples.

2.3. Association of alleles with MS subtypes and disability status Allele association of the genotyped markers in patients with inflammatory MS, progressive MS (including SPMS and PPMS) and the whole CDMS group (group including all MS patients) was assessed. The association of alleles with neurological disability expressed by EDSS scores and progression index was analyzed in both inflammatory and progressive MS groups.

2.4. Association of haplotypes with MS subtypes and disability Haploview program (http://www.broadinstitute.org/scientificcommunity/science/programs/medical-and-population-genetics/haplo view/haploview)wasusedtoexaminelinkagedisequilibriumpatternsand likelyhaplotypefrequencies.Thelinkagedisequilibrium(LD)ateachofthe three loci (TPH1, TPH2, MTNR1B) was studied using data from the Hapmap phase III from Feb09 (CEU population, assembly build36, found at http://hapmap.ncbi.nlm.nih.gov). Visual inspection of loci gave an idea of the overall LD patterns, and individual D' values were obtained by analyzing the Hapmap data in Haploview 4.2. The two markers within TPH1 (rs1800532, rs10488682), TPH2 (rs4570625, rs10506645) and MTNR1B (rs4753426, rs10830963) were each in strong linkage disequilibrium (D' > 0.5, data not shown) and were used in haplotype analyses. Haplotype association analysis was performed in different subtypes, group including all MS patients and controls. The association of haplotypes with gender and EDSS scores was also determined.

2.5. Statistical analyses Among the seven SNPs studied, six SNPs (rs1800532, 10488982, rs10506645, rs4570625, rs4753426 and rs10830963) had a minor allele frequency (MAF) above 2%, while one SNP, rs12942767 had a very low MAF and most individuals were homozygous for the G allele. Allele and haplotype associations in controls and in different subtypes of MS patients were analyzed using Chi-square (χ 2) analysis, implemented in the PASW (version 18.0, SPSS Inc., Chicago, Illinois, USA) and the Haploview program. The association between alleles and the progression index in the inflammatory or progressive subtypes of MS was analyzed with Mann–Whitney U test. Due to the explorative nature of this study, p b 0.05 was considered significant.

3. Results The demographic and clinical characteristics of the subjects including age, gender, disease duration, EDSS scores and progression index are summarized in Table 1. As expected, the patients in the SPMS and PPMS groups were older and had longer disease duration (DD) and higher EDSS scores than the patients in the RRMS [DD years (mean ± SD): SPMS (12.8 ± 7.0), PPMS (8.4 ± 7.1) vs RRMS (6.1 ± 5.2); EDSS (mean ± SD): SPMS (5.3 ± 1.4), PPMS (4.8 ± 2.3) vs RRMS (2.0 ± 2.0)].

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Table 1 Demographic and clinical characteristics of MS patients and controls. Characteristics

Number of patientsc Sex (male/female)c Age (years)d Duration of disease (DD) (years)d EDSSd Progression Index (PI)d (EDSS/DD)

Inflammatory MSa

Progressive MSb

RRMS

SPMS

PPMS

109

51

33

397

28/81 37.3(± 10.1) 6.1(±5.2)

21/30 47.4(± 9.0) 12.8(± 7.0)

18/15 51.8(±10.3) 8.4(±7.1)

216/181 44.3(±11.1) –

2.0 (± 2.0) 0.63(± 0.92)

5.3 (± 1.4) 0.57(± 0.40)

4.8 (± 2.3) 0.75(±1.00)

– –

Controls

MS Multiple Sclerosis, RRMS Relapsing Remitting Multiple Sclerosis, SPMS Secondary Progressive Multiple Sclerosis, PPMS Primary Progressive Multiple Sclerosis. a Inflammatory MS group (RRMS subtype). b Progressive MS group (Progressive subtypes, SPMS and PPMS). c Number of patients. d Mean ± SD.

3.1. Association of TPH2 markers with MS subtypes and neurological disability

were not detected, but the decreased C allele frequency tended to associate with rs10830963 in the progressive MS group (Table 3). Significant differences in genotype distribution between the subtypes were not seen. Likewise, there was no allelic association between the two MTNR1B SNPs with disability status or disease progression index (data not shown). Association of MTNR1B haplotypes with disease susceptibility and disability status was analyzed further (Tables 2–4). The rs10830963-rs4753426 G-T haplotype was associated with the risk of MS in the progressive MS group (p b 0.05). Among the progressive subtypes, this haplotype tended to associate with disease risk in PPMS (p = 0.057). However, in the SPMS group, another haplotype, rs10830963-rs4753426 G-C was associated with the risk of disease (p b 0.05). Analyses of gender-based association of the rs10830963rs4753426 G-T haplotype in the progressive MS group, revealed an association of this haplotype with the risk of MS in males with SPMS (p b 0.05). No association was found between the TPH1 markers (rs1800532 and 10488982) or AANAT marker (rs12942767) and the risk of MS in any of the phenotypes studied. 4. Discussion

As indicated in Table 3, the increased T allele frequency of SNP rs4570625 in the transcriptional control region of the TPH2 gene was associated with an increased risk of MS in the progressive MS subtypes (pb 0.05) [Odds ratio (95% confidence interval): 1.665 (1.023–2.709)]. There was no significant difference in genotype distribution between the subtypes (data not shown). Likewise, no allelic association between the two TPH2 SNPs and disability or disease progression was found. The association of TPH2 marker haplotypes with the disease subtypes and disability status was studied further (Tables 2–4). In males with progressive MS, the rs4570625-rs10506645 T-C haplotype was associated with an increased risk of MS (p b 0.05), and this effect was mainly due to the males of the SPMS group (p b 0.05). When looking at the association of TPH2 haplotypes with EDSS scores, it appeared that in PPMS, the T-T haplotype was significantly associated with the risk of disability in more severely disabled group (EDSS score >4) (p b 0.05), while the T-C haplotype was associated with milder disability in SPMS group (EDSS b 4) (p b 0.05). 3.2. Association of MTNR1B markers with MS subtypes and disability Significant allelic associations of the SNPs rs4753426 and rs10830963 in MTNR1B gene with MS or any of its phenotypes

In this exploratory study we addressed the relationship between melatonin pathway and MS disease by analyzing the polymorphisms of melatonin pathway genes in different subtypes of MS and determined their association with neurological disability. Our results showed that the SNPs in the TPH2 and MTNR1B genes involved in melatonin biosynthesis and function are associated with the risk of MS and more severe disability among patients with progressive subtypes of MS. In the present study we were able to show an association of the SNPs, rs4570625 and rs10506645, with the risk of disability status in the progressive MS subtypes. These SNPs have been shown to be associated with cognitive decline and psychiatric disorders (Mossner et al., 2006; Campos et al., 2011). In the present study, we identified an increased T allele frequency of the SNP rs4570625 (−703 G/T) in the promoter region of TPH2 gene in progressive MS subtypes. Earlier studies have also noticed the increased T allele frequency of this SNP in patients with executive dysfunction (Reuter et al., 2007; Osinsky et al., 2009) and attention deficits (Leppanen et al., 2011). Additionally, in the progressive MS subtypes we observed that the haplotypes of the TPH2 SNPs were associated with the risk of severe disability. The haplotype rs4570625rs10506645TT of TPH2 gene was associated with the risk of severe

Table 2 Haplotype association of polymorphisms within the TPH2 and MTNR1B genes in progressive MS group and its subtypes compared to healthy controls. Progressive MSa (n = 84)

SPMS (n = 51)

PPMS (n = 33)

Haplotypes

Case/control frequency

χ2

p-value

Case/control frequency

χ2

p-value

Case/control frequency

χ2

p-value

TPH2/rs4570625 GC TT GT TC

(G/T) – rs10506645 (C/T) 0.735/0.786 0.206/0.149 0.035/0.047 0.024/0.018

2.191 3.446 0.413 0.255

0.139 0.063⁎ 0.520 0.613

0.749/0.786 0.194/0.149 0.029/0.047 0.029/0.018

0.773 1.452 0.718 0.548

0.379 0.228 0.397 0.459

0.711/0.786 0.226/0.149 0.047/0.047 0.016/0.018

2.019 2.772 0.002 0.011

0.155 0.096⁎ 1.0 0.915

0.275 0.261 0.376 0.027⁎⁎

0.335/0.411 0.409/0.311 0.202/0.251 0.054/0.026

2.28 4.125 1.237 2.5

0.131 0.042⁎⁎ 0.266 0.114

0.417/0.411 0.268/0.311 0.247/0.251 0.068/0.026

0.0090 0.543 0.0050 3.627

0.924 0.461 0.945 0.057⁎

MTNR1B/rs10830963 (C/G) – rs4753426 (C/T) CT 0.366/0.411 1.19 GC 0.355/0.311 1.265 CC 0.219/0.251 0.783 GT 0.059/0.026 4.875

MS Multiple Sclerosis, SPMS Secondary Progressive Multiple Sclerosis, PPMS Primary Progressive Multiple Sclerosis. Significant values are in bold. a Progressive MS group (Progressive subtypes, SPMS and PPMS subtypes). ⁎⁎ p value b 0.05. ⁎ p value b 0.10 (tendency of association).

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Table 3 Association of haplotypes of polymorphisms in TPH2 and MTNR1B genes in males and females with progressive MS group and its subtypes compared to controls. Progressive MSa (n = 84)

SPMS (n = 51) Case/control frequency

χ2

p-value

Case/control frequency

χ2

p-value

0.664 0.071⁎ 0.167 0.196

0.780/0.781 0.202/0.139 0.017/0.059 0.001/0.021

0.0 1.67 1.922 1.188

0.99 0.196 0.166 0.276

0.712/0.781 0.248/0.139 0.038/0.059 0.002/0.021

0.691 2.45 0.222 0.499

0.406 0.117 0.638 0.48

Males: TPH2/rs4570625 (G/T) – rs10506645 (C/T) GC 0.724/0.792 1.758 TT 0.187/0.157 0.439 GT 0.038/0.036 0.01 TC 0.051/0.016 3.403

0.185 0.507 0.921 0.047⁎⁎

0.738/0.792 0.166/0.157 0.024/0.036 0.072/0.016

0.657 0.024 0.152 5.922

0.417 0.877 0.696 0.015⁎⁎

0.710/0.792 0.210/0.157 0.053/0.036 0.027/0.016

1.373 0.713 0.299 0.279

0.241 0.398 0.585 0.5971

Females: MTNR1B/rs10830963 (C/G) – rs4753426 (C/T) CT 0.354/0.438 2.075 GC 0.356/0.318 0.474 CC 0.242/0.217 0.258 GT 0.048/0.027 1.025

0.150 0.491 0.611 0.311

0.322/0.438 0.432/0.318 0.209/0.217 0.037/0.027

2.94 3.117 0.02 0.181

0.086 0.077⁎ 0.886 0.670

0.427/0.438 0.184/0.318 0.316/0.217 0.073/0.027

0.012 2.21 1.479 1.849

0.912 0.137 0.224 0.174

Males: MTNR1B/rs10830963 (C/G) – rs4753426 (C/T) CT 0.376/0.388 0.044 GC 0.363/0.305 1.052 CC 0.187/0.281 3.053 GT 0.074/0.026 4.858

0.834 0.305 0.081⁎ 0.027⁎⁎

0.346/0.388 0.393/0.305 0.178/0.281 0.083/0.026

0.288 1.389 2.052 4.107

0.591 0.239 0.152 0.043⁎⁎

0.408/0.388 0.330/0.305 0.197/0.281 0.065/0.026

0.061 0.1 1.248 1.906

0.805 0.752 0.264 0.167

Case/control frequency

Females: TPH2/rs4570625 (G/T) – rs10506645 GC 0.759/0.781 TT 0.216/0.139 GT 0.023/0.059 TC 0.002/0.021

χ2

PPMS (n = 33)

p-value

Haplotypes

(C/T) 0.189 3.264 1.908 1.67

MS Multiple Sclerosis, SPMS Secondary Progressive Multiple Sclerosis, PPMS Primary Progressive Multiple Sclerosis. Significant values are in bold. a Progressive MS group (SPMS and PPMS subtypes). ⁎⁎ p‐value b 0.05. ⁎ p‐value b 0.10 (tendency of association).

disability in primary progressive MS (PPMS), while haplotype rs4570625-rs10506645TC appeared to be protective against disability in secondary progressive MS (SPMS). This clearly shows that the T allele of rs4570625 is the risk allele of neurological disability, while the C/T allele of rs10506645 seems to contribute to the disease type in modulating its effect. Taken together, these observations suggest that there could be at least two TPH2 SNP variants that interact together in modulating the risk towards disability in MS. It may be that the presence of this polymorphism rs4570625 (−703 G/T) in the promoter region affects the serotonin biosynthesis in melatonin pathway of progressive MS patients thereby influencing the neurological functions in such individuals. MTNR1B gene in chromosome 11q21-22 encodes for a receptor which mediates several functions of melatonin (Dubocovich et al., 2003). The haplotype analysis in this study revealed a significant

association of rs10830963-rs4753426 haplotype G-T with the progressive MS group including both SPMS and PPMS, while the SPMS possessed haplotype rs10830963-rs4753426 G-C. This suggests that the G allele of rs10830963 is a risk allele for the progressive forms of MS. However, the involvement of T/C allele of rs4753426 in the risk of progressive disease should also be accounted for. Recently it has been shown that the C allele of rs4753426 is associated with decreased sunshine duration per day (Ji et al., 2010). Although we did not observe any striking difference between the alleles of rs4753426, the increased frequency of C allele seen in our patients suggests their adaptability to existing dark environmental conditions. Consistent with our observation, the selection of derived rs4753426C allele is believed to occur in order to adapt individuals to environmental pressure and may also increase the immunostimulatory effect of melatonin (Ji et al., 2010).

Table 4 Association of haplotypes of polymorphisms in TPH2 and MTNR1B genes with disability status in progressive MS group and its subtypes. Progressive MSa (n = 84) Haplotypes

Low/High EDSS frequency

TPH2/rs4570625 (G/T) – rs10506645 (C/T) GC 0.717/0.772 TT 0.233/0.136 GT 0.025/0.069 TC 0.025/0.023 MTNR1B/rs10830963 CT GC CC GT

(C/G) – rs4753426 (C/T) 0.340/0.396 0.366/0.282 0.239/0.218 0.055/0.104

SPMS (n = 51)

PPMS (n = 33)

p-value

Low/High EDSS frequency

p-value

Low/High EDSS frequency

p-value

0.476 0.173 0.187 0.942

0.755/0.699 0.194/0.199 0.038/0.001 0.013/0.101

0.608 0.958 0.388 0.040⁎⁎

0.642/ 0.308/ 0.049/ 0.001/

0.833 0.083 0.042 0.042

0.099 0.035⁎⁎ 0.901 0.202

0.505 0.317 0.772 0.269

0.401/0.395 0.328/0.295 0.221/0.205 0.050/0.105

0.963 0.781 0.874 0.362

0.364/ 0.279/ 0.292/ 0.064/

0.471 0.238 0.179 0.113

0.396 0.713 0.308 0.491

MS Multiple Sclerosis, SPMS Secondary Progressive Multiple Sclerosis, PPMS Primary Progressive Multiple Sclerosis, EDSS Expanded disability Status Scale. Low EDSS group is mildly disabled (EDSS b 4). High EDSS group is more severely disabled (EDSS > 4). Significant values are in bold. a Progressive MS group (SPMS and PPMS subtypes). ⁎⁎ p‐value b 0.05.

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In conclusion, this exploratory study suggests an involvement of melatonin pathway in the MS. The polymorphisms in TPH2 and MTNR1B genes may interact together in modulating the risk of the disease and accumulation of disability in the progressive subtypes of MS. The association of alleles and haplotypes of the promoter SNP in the TPH2 (rs4570625) gene and the SNPs rs4753426 and rs10830963 in the MTNR1B gene implies the dysregulation in melatonin pathway in these progressive patients which may facilitate the accumulation of neurological disability. In order to confirm these preliminary data, additional studies with larger sample size and functional experiments covering the entire gene variability of the studied genes are needed. Our data suggesting the contribution of melatonin to MS susceptibility and disability could provide a rationale for future clinical trials on the effects of melatonin in overcoming neurodegeneration in MS. Acknowledgments We thank bioanalyst Raija Paalavuo, Neuroimmunology Unit, University of Tampere for technical assistance, Heini Huhtala, PhD School of Health Sciences for her advice in statistical analyses and bioanalyst Pauli Mäkinen, Department of Clinical Medicine, for his excellent technical assistance. The study was financially supported by Competitive Research Funding of Tampere University Hospital (9 M048 and 9 N035). Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.jneuroim.2012.05.014. References Akpinar, Z., Tokgoz, S., Gokbel, H., Okudan, N., Uguz, F., Yilmaz, G., 2008. The association of nocturnal serum melatonin levels with major depression in patients with acute multiple sclerosis. Psychiatry Res. 161, 253–257. Campos, S.B., Miranda, D.M., Souza, B.R., Pereira, P.A., Neves, F.S., Tramontina, J., Kapczinski, F., Romano-Silva, M.A., Correa, H., 2011. Association study of tryptophan hydroxylase 2 gene polymorphisms in bipolar disorder patients with panic disorder comorbidity. Psychiatr. Genet. 21, 106–111. Chattoraj, A., Liu, T., Zhang, L.S., Huang, Z., Borjigin, J., 2009. Melatonin formation in mammals: in vivo perspectives. Rev. Endocr. Metab. Disord. 10, 237–243. Dubocovich, M.L., Rivera-Bermudez, M.A., Gerdin, M.J., Masana, M.I., 2003. Molecular pharmacology, regulation and function of mammalian melatonin receptors. Front. Biosci. 8, d1093–d1108. Hafler, D.A., 2004. Multiple sclerosis. J. Clin. Invest. 113, 788–794. Hauser, S.L., Oksenberg, J.R., 2006. The neurobiology of multiple sclerosis: genes, inflammation, and neurodegeneration. Neuron 52, 61–76.

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